Published January 20, 2004
JCB
Article
Trans-SNARE interactions elicit Ca2! efflux
from the yeast vacuole lumen
Alexey J. Merz and William T. Wickner
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755
Vacuoles lacking either of two complementary SNAREs,
Vam3p or Nyv1p, fail to release Ca2! upon tethering. Mixing
these two vacuole populations together allows Vam3p
and Nyv1p to interact in trans and rescues Ca2! release.
Sec17/18p promote sustained Ca2! release by recycling
SNAREs (and perhaps other limiting factors), but are not
required at the release step itself. We conclude that transSNARE assembly events during docking promote Ca2!
release from the vacuole lumen.
Introduction
Conserved molecules govern membrane docking and fusion
in the secretory and endocytic pathways of eukaryotic cells
(Jahn et al., 2003). These molecules include Rab/Ypt
GTPases, Sec17/18p (NSF/"-SNAP) chaperones, Sec1p/
Munc18 (SM) proteins, and SNARE proteins. SNAREs fold
into tight core complexes containing four coiled " helices
that are contributed by three or four SNARE proteins
(Sutton et al., 1998; Jahn et al., 2003). SNAREs are categorized
as Q or R, based on whether they contribute a glutamine or
arginine to the ionic (zero) layer of the SNARE core complex
(Fasshauer et al., 1998). Core complexes typically contain
three Q-SNARE coils and an R-SNARE coil. Complementary
SNAREs must be present in trans on docked, apposed
membranes for fusion to occur (Nichols et al., 1997).
Trans-SNARE complexes are hypothesized to assume a
“SNAREpin” structure (Weber et al., 1998; Jahn et al.,
2003) similar to the tetrahelical SNARE core complex, but
with the SNARE transmembrane anchors embedded in
opposite docked membranes. The exact functions of transSNARE complexes are unresolved (Jahn et al., 2003).
Cytosolic Ca2! is required for many membrane fusion
events. In some cases, ambient [Ca2!] is sufficient for fusion
(Beckers and Balch, 1989; Baker et al., 1990); in other cases,
The online version of this paper contains supplemental material.
Address correspondence to William T. Wickner, Dept. of Biochemistry,
7200 Vail Bldg., Dartmouth Medical School, Hanover, NH 03755-3844.
Tel.: (603) 650-1701. Fax: (603) 650-1353.
email: [email protected]
Key words: membrane; docking; fusion; Rab GTPase; SM-protein
 The Rockefeller University Press, 0021-9525/2004/01/195/12 $8.00
The Journal of Cell Biology, Volume 164, Number 2, January 19, 2004 195–206
http://www.jcb.org/cgi/doi/10.1083/jcb.200310105
transient increases in [Ca2!] are needed (Sullivan et al.,
1993; Peters and Mayer, 1998; Chen et al., 1999; Pryor et
al., 2000; Reddy et al., 2001; Rettig and Neher, 2002). In
neurons, depolarization opens voltage-gated Ca2! channels,
triggering exocytosis (Rettig and Neher, 2002; Jahn et al.,
2003). However, little is known about how Ca2! regulates
intracellular fusion events such as endosome–endosome fusion.
Even less is known about how Ca2! signals that regulate intracellular membrane fusion are themselves regulated. Ca2!
channels in the plasma membranes of mammalian cells
physically and functionally interact with the Q-SNAREs
syntaxin and SNAP-25 (Bennett et al., 1992; Yoshida et al.,
1992; Sheng et al., 1994; Mochida et al., 1996; Wiser et al.,
1996; Rettig et al., 1997). Ca2! channels also interact with
the exocytic machinery via the Sec6/8 complex and Rab3interacting molecule binding proteins (Shin et al., 2000;
Hibino et al., 2002). On the yeast vacuole, the R-SNARE
Nyv1p inhibits the vacuolar Ca2! ATPase Pmc1p (Takita et
al., 2001). It is unclear how such interactions are integrated
into cycles of membrane docking and fusion.
Trans interactions between SNARES on opposite membranes have been proposed to facilitate or trigger Ca 2!
signals in response to docking (Bezprozvanny et al., 1995;
Wiser et al., 1996; Schekman, 1998), but technical obstacles
have precluded direct tests of this hypothesis. The yeast
vacuole offers powerful genetic and biochemical tools that
Abbreviations used in this paper: GDI, GDP dissociation inhibitor; RDI,
Rho GDI; rVam7p, recombinant Vam7p; SM, Sec1p/Munc18.
Supplemental Material can be found at:
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The Journal of Cell Biology
C
a2! transients trigger many SNARE-dependent membrane fusion events. The homotypic fusion of yeast
vacuoles occurs after a release of lumenal Ca2!.
Here, we show that trans-SNARE interactions promote the
release of Ca2! from the vacuole lumen. Ypt7p–GTP, the
Sec1p/Munc18-protein Vps33p, and Rho GTPases, all of
which function during docking, are required for Ca2! release.
Inhibitors of SNARE function prevent Ca2! release. Recombinant Vam7p, a soluble Q-SNARE, stimulates Ca2! release.
Published January 20, 2004
196 The Journal of Cell Biology | Volume 164, Number 2, 2004
allow trans-SNARE interactions to be manipulated in a
physiologically relevant in vitro docking and fusion reaction.
Vacuole fusion begins with priming. In this step, Sec17/18p
(yeast "-SNAP/NSF) consumes ATP to disassemble cisSNARE complexes (Mayer et al., 1996; Ungermann et al.,
1998a). In the next stage, docking, the Rab GTPase Ypt7p
promotes membrane tethering (Mayer and Wickner, 1997;
Ungermann et al., 1998b), and specialized subdomains
called vertex sites assemble at the docking junction (Wang et
al., 2002, 2003). During Ypt7p-mediated docking, Ca2! is
released from the vacuole lumen (Peters and Mayer, 1998;
Eitzen et al., 2000). This Ca2! signal is necessary for fusion
because depletion of lumenal Ca2! makes fusion dependent
on added Ca2!, and chelation of extralumenal Ca2! with
BAPTA prevents fusion (Peters and Mayer, 1998). We now
show that Ca2! release during docking occurs in response to
trans-SNARE interactions.
Results
Figure 1. Characterization of rVam7p and Vam7p antagonists.
(A) Stimulation of fusion by rVam7p. Standard fusion reactions
(see Materials and methods) were initiated in the presence of
rVam7p at the indicated concentrations, with or without 10 nM
of Sec18p. “Ice” indicates fusion signal in a control reaction
incubated on ice instead of at 27#C. (B) Inhibition of fusion by
rVam7p PX domain. (C) Inhibition of fusion by affinity-purified
anti-Vam7p antibody. “Boiled Ab” indicates a sample containing
anti-Vam7p heat denatured by incubation for 5 min at 100#C.
(D) rVam7p reverses a fusion block imposed by 150 nM of affinitypurified anti-Vam7p antibody. “No Ab” indicates control samples
without antibody. (E and F) Reversal of a PX block by rVam7p and
characterization of the block. In E, reactions were initiated in the
presence of 20 $M of PX domain and incubated for 30 min at 27#C,
and the indicated inhibitors were added. 4 min later, 10 $M of
rVam7p was added to reverse the PX block; the reactions were
incubated at 27#C for an additional 60 min. Reactions moved to
ice before rescue (ice), or not rescued by rVam7p (no rescue),
were strongly inhibited by PX domain. In F, the indicated inhibitors
were added from the beginning of the fusion reaction. Affinity-purified
anti-Vam3p antibody was added at 58, 115, 230, or 460 nM.
is in an altered conformational or oligomeric state at the end
of the PX block. Strikingly, partial resistance to the Ca2! chelator BAPTA was also observed (Fig. 1 E). This BAPTA resistance was surprising for two reasons. First, although BAPTA
inhibits a late stage in homotypic vacuole fusion, Vam7p was
previously assigned to the intermediate docking stage in kinetic assays (Ungermann and Wickner, 1998; Boeddinghaus
et al., 2002). Second, staging experiments had shown that the
vacuole fusion reaction reaches a PX, or anti-Vam7p, resistant state in the presence of BAPTA, suggesting that Vam7p
function is completed before Ca2! action, not after (Boeddinghaus et al., 2002). Two possible explanations for these
apparently divergent results are that the added rVam7p increases the number or sensitivity of Ca2! sensors, or that it in-
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Vam7p stimulates fusion and reduces its BAPTA sensitivity
We were led to explore the roles of SNAREs in Ca2! flux by
experiments with the soluble Q-SNARE Vam7p. During
priming, Sec17/18p disassembles cis-SNARE complexes.
Vam7p, having neither a hydrophobic transmembrane anchor nor a covalently attached lipid anchor, is released from
the membrane during priming (Ungermann and Wickner,
1998; Ungermann et al., 1998a; Boeddinghaus et al., 2002).
For fusion to occur, Vam7p must reassociate with the vacuole membrane via its amino-terminal PX domain, which
binds to phosphatidylinositol-3–phosphate (Cheever et al.,
2001; Boeddinghaus et al., 2002). We found that recombinant Vam7p (rVam7p) promoted fusion in vitro (Fig. 1 A).
This stimulation was more pronounced when recombinant
Sec18p was not added to the reaction (Fig. 1 A), probably
because added Sec18p promotes the release of endogenous
Vam7p from SNARE complexes on the vacuole. Recombinant PX domain prevented Vam7p reassociation (Boeddinghaus et al., 2002) and hence fusion (Fig. 1 B). Anti-Vam7p
antibody (Fig. 1 C) also prevented fusion (Ungermann and
Wickner, 1998; Boeddinghaus et al., 2002). rVam7p reversed the PX and anti-Vam7p fusion blocks when added
either at the beginning of the reaction (Fig. 1 D; PX, not
depicted) or 30 min into the reaction, after priming
and during docking (Fig. 1 E; anti-Vam7p, not depicted).
rVam7p did not rescue fusion in reactions inhibited by antibodies against the SNARE Vam3p (Fig. 1, D and E), verifying that rVam7p specifically reverses blockades of Vam7p
function.
Next, we asked whether various inhibitors of fusion (Fig. 1
F) acted before, during, or after the step(s) prevented by PX
domain (Fig. 1 E). Fusion reactions were initiated in the presence of PX domain and incubated for 30 min to allow priming and docking, then the PX block was reversed with
rVam7p. During the PX blockade, the reaction passed
through the priming stage, as shown by the acquisition of resistance to anti-Sec17p antibody (Fig. 1 E). Anti-Vam3p antibody, which in kinetic experiments inhibits at docking
(Nichols et al., 1997), inhibited the reaction at high but not
low concentrations (Fig. 1 E). This may indicate that Vam3p
Published January 20, 2004
Ca2! and SNAREs | Merz and Wickner 197
Figure 2. Docking-dependent Ca2! release depends on Ypt7p–GTP, Rho GTPases, and the SM-protein Vps33p. Standard Ca2! release reactions
(see Materials and methods) were initiated in the absence of inhibitors (“standard”; closed circles in each plot) or in the presence of the indicated
reagents: (A) 380 nM of anti-Sec17p IgG, 2.5 $M of GDI, or 100 nM of affinity-purified anti-Vps33p; (B) 50 $M of Gyp1–46; (C) 15 $M of
RDI. (A–C) Closed circles denote standard reaction conditions. In A, open squares denote anti-Sec17p; open triangles denote Rab GDI; open
inverted triangles denote anti-Ypt7p; and open circles denote anti-Vps33p. In B, open circles denote Gyp1-46 treatment. In C, open circles
denote Rho GDI (RDI) treatment.
creases the frequency or amplitude of Ca2! efflux events, resulting in reduced BAPTA sensitivity.
Figure 3. SNAREs promote docking-dependent Ca2!
release. Standard reactions contained: (A) 150 nM of
affinity-purified anti-Vam7p and/or 10 $M of Vam7p;
(B) 20 $M of PX domain and/or 10 $M of Vam7p; (C) 120
nM of affinity-purified anti-Nyv1p or 200 nM of affinitypurified anti-Vam3p. (D) 1.6 $M of affinity-purified
anti-Vti1p or 120 nM of affinity-purified anti-Nyv1p.
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Docking factors and SNAREs promote Ca2! release
To see if Vam7p influences Ca2! efflux from the vacuole lumen, we used the photoprotein aequorin to monitor the extralumenal Ca2! concentration [Ca2!]E during in vitro
docking and fusion (Peters and Mayer, 1998). We began by
evaluating the importance of proteins that function during
docking.
Vacuoles actively sequester extralumenal Ca2! during the
first minutes of incubation with ATP (Fig. 2 A, standard).
Experiments with the slow Ca2! chelator EGTA indicate
that the relatively high initial [Ca2!]E is due to ambient
Ca2! present in buffer solutions, and is not necessary for
docking or fusion (Peters and Mayer, 1998; unpublished
data; Margolis, N., personal communication). During docking, Ca2! is released from the vacuole lumen into the extralumenal space, with [Ca2!]E typically peaking at 30–40
min (Fig. 2 A). As reported previously (Peters and Mayer,
1998), this Ca2! release requires both Sec17/18p-mediated
priming and Ypt7p-mediated docking. Ca2! was sequestered
in the vacuole lumen but not released in the presence of
anti-Sec17p antibody (Fig. 1 A). Recombinant Gdi1p
(GDI; GDP dissociation inhibitor [GDI]) or affinity-purified anti-Ypt7p antibody, both inhibitors of Ypt7p function,
also prevented Ca2! release (Fig. 2 A).
To determine whether the GTP-bound form of Ypt7p is
needed for Ca2! release, we used Gyp1–46p and Gyp7–47p,
GTPase activating proteins (GAPs) that promote the hydrolysis of Ypt7p-bound GTP (Vollmer et al., 1999; Eitzen et
al., 2000). These GAPs prevent SNAREs and other fusion
factors from accumulating at “vertex” membrane subdomains during docking and prevent fusion (Eitzen et al.,
2000; Wang et al., 2003). Both Gyp1–46p (Fig. 2 B) and
Gyp7–47p (not depicted) prevented Ca2! release, indicating
a requirement for Ypt7p–GTP. The Rho GTPases Cdc42p
and Rho1p also function during docking (Eitzen et al.,
2001; Muller et al., 2001), possibly by promoting actin remodeling on the vacuole membrane (Eitzen et al., 2002).
Rho GDI (RDI) extracts Rho GTPases from membranes
and prevents fusion (Eitzen et al., 2001). RDI prevented
Ca2! release during docking (Fig. 2 C). The SM protein
Vps33p, a component of the HOPS–class C docking complex (Seals et al., 2000), functions during docking and regulates SNARE complex formation (Price et al., 2000; Sato et
al., 2000). Anti-Vps33p antibody prevented Ca2! efflux
(Fig. 2 A). Thus docking-dependent Ca2! release from the
vacuole lumen requires Ypt7p–GTP, vacuolar Rho GTPases, and the SM protein Vps33p.
When rVam7p was added to a standard fusion assay, it
caused an increase in docking-dependent Ca2! efflux (Fig.
3 A). Ca2! release was blocked by affinity-purified antiVam7p antibody or PX domain (Fig. 3, A and B). rVam7p
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198 The Journal of Cell Biology | Volume 164, Number 2, 2004
Figure 4. Trans-SNARE interactions elicit dockingdependent Ca2! release. The data plotted in A–D are
from the same assay. Standard reactions contained:
(A) 10 $g (protein content) of vacuoles derived from
the parental strain BJ3505; (B) 10 $g of vacuoles from
an nyv1% derivative of BJ3505; (C) 10 $g of vacuoles
from a vam3% derivative of BJ3505; (D) a mixture of 5 $g
each of vacuoles from the vam3% and nyv1% strains.
Symbols: closed circles, standard reaction conditions;
open circles, reactions with recombinant GDI; triangles,
reactions with anti-Vam3p antibody; inverted triangles,
reactions with anti-Nyv1p antibody. Inhibitor concentrations are as in Fig. 1. With Nyv1p-deficient vacuoles,
a small but reproducible decrease in [Ca2!]E was observed
in the presence of docking inhibitors or anti-SNARE
antibodies. This residual docking-dependent Ca2! release
(&5% of that seen with vacuoles from wild-type cells)
may be mediated by R-SNAREs other than Nyv1p.
Trans-SNARE interactions elicit Ca2! release
Next, we asked whether vacuoles lacking either of two
SNARE proteins would exhibit Ca2! efflux upon Ypt7p-mediated tethering. Vacuoles obtained from cells bearing
nyv1% or vam3% null mutations (Fig. 4, B and C) took up
Ca2! from the extralumenal space but failed to release it. In
reactions containing vacuoles that lacked either Nyv1p or
Vam3p, [Ca2!]E was unresponsive to recombinant Gdi1p
(Fig. 4, B and C) or anti-Ypt7p antibody (not depicted).
Similarly, these SNARE-deficient vacuoles were unresponsive to antibodies against Vam3p, Nyv1p (Fig. 4, B and C)
or Vam7p (not depicted). Both Nyv1p and Vam3p-deficient vacuoles undergo Ypt7p-dependent tethering (Ungermann et al., 1998b; Wang et al., 2003). The severe Ca 2!
release defects exhibited by these vacuoles shows that membrane tethering, without a full complement of SNAREs, is
not sufficient to trigger Ca2! release.
Vam3p and Nyv1p can promote fusion from opposite
membranes, i.e., in trans (Nichols et al., 1997; Ungermann
et al., 1998b). Vacuoles lacking Vam3p do not fuse with
vacuoles lacking Vam3p, and vacuoles lacking Nyv1p do not
fuse with vacuoles lacking Nyv1p. However, vacuoles lacking Vam3p do fuse with vacuoles lacking Nyv1p, indicating
that these Q- and R-SNAREs functionally interact in trans
(Nichols et al., 1997; Ungermann et al., 1998b). We therefore asked whether docking-dependent Ca2! flux is also restored by the mixture of Vam3p- and Nyv1p-deficient vacuoles. As shown in Fig. 4 D, this was indeed the case. Ca2!
release by the vacuole mixture was docking-dependent because it was prevented by GDI blockade of Ypt7p function.
Ca2! release by mixtures of vacuoles from vam3% and
nyv1% cells is blocked by antibodies against Vam3p or
Nyv1p (Fig. 4 D). Thus, functional trans interactions between Q- and R-SNAREs trigger docking-dependent Ca2!
release from the vacuole lumen.
Cycles of trans-SNARE complex assembly promote
Ca2! release
Sec17/18p disassemble complexed SNARE proteins. Recombinant Sec17p and Sec18p, and antibodies against these
proteins, have provided useful tools for the manipulation of
SNARE assembly dynamics. To further test whether the oligomeric state of SNARE proteins regulates Ca2! release, we
tested the effect of recombinant Sec18p (Fig. 5 A). Sec18p
stimulated Ca2! release at up to 300 nM; at higher concentrations this stimulation declined (see Discussion). The
stimulatory effect of rVam7p and Sec18p (Fig. 5 A) combined was greater than with either reagent alone.
Next, we asked whether the effect of Sec17/18p was confined to the early, priming phase of the reaction. Standard
reactions were started, and various reagents were added to
the reactions at !18 min, after priming (Mayer et al., 1996;
Ungermann et al., 1998a) and during the normal dockingdependent Ca2! efflux (Fig. 5 B). Recombinant Sec18p
caused an immediate increase in the amplitude of Ca2! efflux (compare high Sec18p to standard), suggesting that
Sec18p regulates the amplitude of Ca2! release during docking. Anti-Sec17p antibodies added either alone or immediately before recombinant Sec18p caused a rapid attenuation
of Ca2! release (Fig. 5 B). Therefore, the stimulatory effect
of recombinant Sec18p depends on endogenous Sec17p.
When added in excess over endogenous Sec18p, recombinant Sec17p inhibits fusion, by recapturing unpaired
SNAREs into cis complexes (Wang et al., 2000). This inhibition of fusion can be reversed by the addition of stoichiometric amounts of Sec18p. Excess Sec17p added at !18
min inhibited Ca2! release (Fig. 5 B), and recombinant
Sec18p reversed this inhibition. Sec18p-mediated stimulation of Ca2! efflux is abolished by anti-Vam3p antibody
(unpublished data), indicating a SNARE requirement. Previous work in our laboratory (Mayer et al., 1996; Ungermann et al., 1998a) showed that anti-Sec17p or anti-Sec18p
antibodies added after !15 min have little effect on fusion,
indicating that Sec17/18p function early (during priming)
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reversed the PX and anti-Vam7p blocks (Fig. 3, A and B).
Ca2! release was also prevented by antibodies against the
Q-SNAREs Vam3p (Fig. 3 C) or Vti1p (Fig. 3 D), or against
the R-SNARE Nyv1p (Fig. 3, C and D). Control IgG, heatdenatured antibodies, and antibodies against the vacuolar
SNARE Ykt6p had no detectable effect on Ca2! release under these conditions (unpublished data). Thus the SNAREs
Vam3p, Vti1p, Vam7p, and Nyv1p regulate docking-dependent Ca2! signaling.
Published January 20, 2004
Ca2! and SNAREs | Merz and Wickner 199
Figure 5. Docking-dependent Ca2! release is stimulated
by Sec17/18p. (A) Simulation of Ca2! release by Sec18p.
(B) Sec17/18p promote ongoing Ca2! release. Standard
reactions were initiated, and at 18 min, the microplate
was removed from the luminometer, and additional
reagents were added: 200 nM of high Sec18p; 150 nM
of high Sec17p; and 380 nM of anti-Sec17p or anti-Sec18p
IgG. Anti-Sec17/18p antibodies were also added from the
reaction start to two samples, as indicated on the plot.
To see if Sec17/18p action and Ca2! release could be uncoupled, we used an antipeptide antibody that permits reversible inhibition of the Ypt7p GTPase. Ypt7p block-reversal can be used to allow Sec17/18p-mediated priming while
accumulating the vacuoles at an early, Ypt7p-dependent
stage of docking. Relief of the anti-Ypt7p block by adding
the antigenic peptide allows the vacuoles to proceed through
docking and fusion in a relatively synchronous manner
(Eitzen et al., 2001, 2002). Reactions were initiated either
Figure 6. Docking-dependent Ca2! release without
ongoing Sec17/18p action. Standard reactions
were initiated in the presence of affinity-purified
anti-Ypt7p peptide antibody and incubated at
26#C. At 30 min, the reactions were moved to ice,
second inhibitors (antibodies against SNAREs or
Sec17p) were added as indicated on the plot,
and the anti-Ypt7p block was reversed by addition
of excess Ypt7p peptide. No peptide was added
to the “no rescue” sample. The samples were then
assayed for Ca2! release at 26#C.
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in a standard fusion reaction. Under the conditions used to
measure Ca2! release similar results were obtained, with fusion inhibited by less than 30% when antibodies were added
at 15–18 min. The observation that Sec17/18p regulate
Ca2! release even after 15 min (during docking) raised the
question of whether Sec17/18p act during the Ca2! release
step itself or act indirectly, by promoting the availability of
unpaired SNAREs or SNARE associated factors such as the
HOPS–class C complex.
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200 The Journal of Cell Biology | Volume 164, Number 2, 2004
Figure 7. Sec17/18p promote sustained Ca2! efflux.
(A) Kinetics of Ca2! release in three block/rescue protocols.
Blocks were imposed in standard reactions for 20 min
before rescue: trace a, anti-Sec18p block/Sec18p reversal;
trace b, anti-Sec17p block/rVam7p reversal; trace c, PX
block/rVam7p reversal. IgG against Sec17p and Sec18p
were used at 380 nM, PX was used at 20 $M, and rVam7p
was used at 10 $M. The dotted line shows the arithmetic
mean of traces a and b. (B) Reactions containing SNAREdeficient vacuoles were initiated in the presence of
anti-Sec17p, incubated for 20 min, and rescued with the
indicated amounts of rVam7p. Control reactions not
rescued with rVam7p (not depicted) did not exhibit Ca2!
efflux, similar to reactions containing both anti-Vam3p
antibody and rVam7p.
First, priming was blocked with anti-Sec18p antibody and
reversed with recombinant Sec18p (Fig. 7 A, trace a). Ca2!
efflux was observed with kinetics similar to those seen in the
standard reaction, with an additional delay corresponding to
the block.
Second, we took advantage of recent observations that,
under appropriate conditions, rVam7p can bypass the requirement for Sec17/18p-mediated priming (unpublished
data; Thorngren, N., personal communication). It appears
that the only essential event mediated by Sec17/18p in vitro
is the liberation of Vam7p from cis-SNARE complexes; sufficient quantities of unpaired integral-membrane SNAREs
are present on unprimed vacuoles to catalyze fusion when
the reaction is supplemented with rVam7p. When rVam7p
was added to an anti-Sec17p-blocked reaction (Fig. 7 A,
trace b), a fast spike of Ca2! release was observed and this
spike rapidly decayed. Similar results were obtained when
rVam7p was used to bypass an anti-Sec18p block (unpublished data). We also asked if rVam7p could interact with
Vam3p and Nyv1p located in trans to one another to promote efflux. For this purpose, a complementation experiment similar to the one shown in Fig. 3 was used. We
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without or with the Ypt7p antibody (Fig. 6). In reactions
blocked with anti-Ypt7p for 35 min and then reversed (Fig.
6), anti-SNARE antibodies added 5 min before reversal were
still inhibitory, indicating that SNARE function is still
needed after priming, and during or after Ypt7p function. In
marked contrast, anti-Sec17p antibodies did not prevent
Ca2! release upon peptide rescue. In a control reaction, antiSec17p, when added from the start of a reaction without
anti-Ypt7p, prevented Ca2! release. Thus, Sec17/18p function is not required during Ca2! release step itself.
We observed consistently that Ca2! release upon reversal
of a Ypt7p block decayed more rapidly in the presence than
in the absence of anti-Sec17p antibody (Fig. 6). This result,
along with the results shown in Fig. 5 B, suggests that
Sec17/18p promotes sustained Ca2! signaling by promoting
cycles of SNARE complex disassembly and trans-complex
formation. To test the idea that Sec17/18p can promote sustained Ca2! release by stimulating SNARE complex turnover and thereby increasing the frequency of trans-SNARE
interactions, we used three different block-reversal protocols
to examine the kinetics of Ca2! efflux (Fig. 7 A). In each
case, the block was maintained for 20 min before reversal.
Published January 20, 2004
Ca2! and SNAREs | Merz and Wickner 201
Figure 8. Fusion triggered by rVam7p. (A) Kinetic window
of anti-Vam3p sensitivity. Standard reactions were initiated in the presence of anti-Sec17p or anti-Sec18p IgG,
incubated for 20 min at 27#C, and rescued with either
Sec18p or Vam7p. At the indicated times after reversal
anti-Vam3p antibody was added to the reactions. Fusion
signals are normalized by subtracting the corresponding
no-rescue signals. (B) Fusion occurs promptly upon
Vam7p rescue of an anti-Sec17p block. Standard reactions
were blocked or not blocked with anti-Sec17p as in A.
Blocked reactions were rescued with Vam7p. Vacuoles
were photographed at the times indicated after rescue,
and membrane surface areas were calculated from vacuole
diameters (see Materials and methods). Bars indicate
geometric mean surface areas with 95% confidence
intervals. Asterisks denote pairs of treatments that differ
at P & 0.001 by the Wilcoxon test. B summarizes data
pooled from two independent experiments that yielded
similar results when analyzed separately.
used to reverse an anti-Sec17p block, we asked if fusion
was accelerated under these conditions. Three approaches
were used. First, we used anti-Vam3p antibody to block
docking at various times after reversal (Fig. 8 A). Within 2
min after reversal, the shortest time tested, fusion had become completely resistant to anti-Vam3p. In contrast,
when anti-Vam3p was added immediately before rVam7p,
no fusion was observed. Thus, Vam3p is required at the
time of rVam7p addition, but it is needed (or its functional
determinants are antibody accessible) for &2 min after
rVam7p addition. To examine fusion more directly, we
measured vacuole size (surface area) under four conditions:
at 1–4 min after Vam7p reversal of anti-Sec17p; at 20–25
min after reversal; in reactions that were blocked but not
reversed; and in reactions not blocked with anti-Sec17p
(Fig. 8 B). We found that the geometric mean surface area
of the vacuoles increased by 54% within 4 min after
rVam7p reversal of the anti-Sec17 block, but no additional
size increase was detected at later times. This methodology
underestimates the actual amount of fusion because some
limiting membrane is lost into the lumen in most fusion
events (Wang et al., 2002), and because vacuoles too small
to resolve (&250-nm diam) likely fuse to form small vacuoles that are then resolved, lowering the population’s measured size. Thus, at least 0.5 round of fusion occurred in
the experiment shown, a result consistent with the amount
of alkaline phosphatase maturation observed in our standard fusion assay (unpublished data). In the third approach, we observed fusion in real time during the 1–4
min interval (Video 1, available at http://www.jcb.org/cgi/
content/full/jcb.200310105/DC1). During this interval,
we observed fusion among !15% of the vacuoles. This
measurement also is likely to underestimate fusion because
recording was initiated !1 min after reversal of the block
by rVam7p, and because events out of the focal plane
could not be clearly resolved. Thus, docking (assayed by
sensitivity to anti-Vam3p), Ca2! efflux, and fusion occur
promptly upon rVam7p-mediated bypass of the priming
block. Together, our data show that the forward pathway
of trans-SNARE complex assembly leads to Ca2! release
and fusion. Sec17/18p are not needed during Ca2! efflux
Downloaded from jcb.rupress.org on April 4, 2011
found that rVam7p elicited transient Ca2! efflux from antiSec17p-blocked mixtures of vacuoles derived from vam3%
and nyv1% cells (Fig. 7 B), but it did not elicit Ca2! efflux
from either population alone. Anti-Vam3p antibody completely suppressed rVam7p rescue (Fig. 7 B). rVam7p bypass
of an anti-Sec17p block thus requires Vam3p and Nyv1p,
even when these SNAREs are available only in trans and not
in cis. These results suggest that on pretethered vacuoles,
rVam7 promotes near-synchronous formation of many
trans-SNARE complexes and rapid Ca2! efflux. In the absence of Sec17/18p activity, rVam7p-elicited efflux is shortlived relative to the sustained release promoted by Sec17/
18p (Fig. 7 A, compare trace a with trace b).
In the third block-reversal protocol, PX domain was used
to block Vam7p reassociation, and rVam7p was used to reverse the PX block as in Fig. 1 F. Under a PX block, priming
and Ypt7p-dependent docking occur, but vertex assembly is
incomplete and trans-SNARE pairing is prevented (Boeddinghaus et al., 2002; Wang et al., 2003). When the PX
block was reversed, an immediate Ca2! spike was observed
(Fig. 7 A, trace c), and this spike was followed by prolonged
Ca2! release. When we plot the average values of the immediate spike from the anti-Sec17p/rVam7p reversal (Fig. 7 A,
trace b) and the sustained but delayed spike from the antiSec18p/Sec18p reversal (Fig. 7 A, trace a), we obtain a curve
(Fig. 7 A, dashed line) that closely matches the rescue curve
for PX/rVam7p reversal. In addition, when the PX block
was reversed by rVam7p in the presence of anti-Sec17p, the
sustained component of the Ca2! flux was absent and only
the spike was observed as in trace b (unpublished data). Together, these experiments show that the reversal of inhibitor
blocks by rVam7p can be used to synchronize Ca2! release,
and confirm that Sec17/18p promotes sustained cycles of
Ca 2! release but need not operate during a single Ca 2!
release event. We conclude that Sec17/18p disassembles
SNARE complexes, producing unpaired SNAREs, which in
turn enter new trans complexes that elicit Ca2! release.
Under standard in vitro conditions (Conradt et al.,
1994; Wang et al., 2002), two to three rounds of fusion
occur in 60–90 min (unpublished data). Because of the
prompt onset and decay of Ca2! release when rVam7p was
Published January 20, 2004
202 The Journal of Cell Biology | Volume 164, Number 2, 2004
but promote sustained Ca2! efflux by catalyzing SNARE
complex turnover and making unpaired SNAREs available
for additional cycles of trans-complex assembly.
Figure 9. SNARE-regulated Ca2! release from vacuoles lacking
Ca2! transporters. Vacuoles isolated from wild-type strain BY4742 or
the indicated isogenic mutant strains (see Materials and methods) were
assayed for Ca2! release in the presence (A) or absence (B) of antiVam3p IgG. Reactions were under standard conditions (see Materials
and methods) except that 250 nM rVam7p was added in all cases. The
variability among the traces in B is comparable to that seen from day to
day among independent vacuole preparations from a single strain.
SNARE dynamics and Ca2! release
A working model of SNARE dynamics and Ca2! function is
shown in Fig. 10. Under standard in vitro fusion conditions,
Sec18/18p-mediated priming (Fig. 10 A) disassembles cisSNARE complexes, yielding unpaired SNAREs on the
membrane and causing the release of endogenous Vam7p.
Docking (Fig. 10 B) entails several steps. Membrane tethering is regulated by the GTPase Ypt7p (Mayer and Wickner,
1997), which also marks the locations where vertex subdomains will be assembled. Vertex assembly is a hierarchical
process that results in the local enrichment of many fusionrelated factors, phosphoinositides and ergosterol (unpublished data; Fratti, R., personal communication), actin, the
HOPS-tethering complex, which includes the SM-protein
Vps33p, and SNAREs (Wang et al., 2002, 2003). Vertex assembly may promote trans-SNARE interactions, which directly or indirectly trigger transient Ca2! release events. Increases in [Ca2!]E in turn promote downstream events (Fig.
10 C) that lead to fusion. Fusion initiates at vertex sites
(Wang et al., 2002); we speculate that both trans-SNARE
pairing and focal Ca2! release occur at the vertex. rVam7p
bypasses the requirement for in vitro Sec17/18p-mediated
priming. When added to tethered vacuoles, Vam7p causes a
rapid, transient spike of Ca2! release and fusion. Sustained
Ca2! release and fusion require ongoing Sec17/18p function
for a continuing supply of unpaired SNAREs.
Trans-SNARE interactions promote Ca2! release
Our experiments with antibodies, rVam7, PX domain, and
vacuoles isolated from SNARE-deficient mutants implicate
four vacuolar SNARE proteins in docking-dependent Ca2!
release: the Q-SNAREs Vam7p, Vam3p, and Vti1p; and
the R-SNARE, Nyv1p. Antibodies against Ykt6p, a fifth
SNARE found in vacuolar cis-complexes (Ungermann et al.,
1999), did not inhibit Ca2! release. As each of these
SNAREs could contribute one coil to a tetrahelical “core
complex” (Sutton et al., 1998), the requirement for all four
is consistent with the hypothesis that tetrahelical transSNARE complexes regulate Ca2! release.
Trans-SNARE interactions can occur only during docking. If Ca2! release is triggered by trans-SNARE interactions, then docking-independent SNARE complex assembly
and disassembly should not cause Ca2! release. Consistent
with this prediction, staging experiments show that SNAREs
are needed for Ca2! release at a post-priming step of the reaction (Figs. 6 and 7). Moreover, the addition of excess
recombinant Sec17p, which inhibits fusion by promoting
the reformation of cis-SNARE complexes from unpaired
SNAREs, attenuates Ca2! release (Fig. 4 C), indicating that
cis-SNARE complex reformation does not trigger Ca2! release. Together, these results indicate that neither cisSNARE complex disassembly nor cis-complex reformation
trigger Ca2! release.
Ypt7p is required for tethering, vertex assembly, and
trans-SNARE pairing (Mayer and Wickner, 1997; Ungermann et al., 1998b; Wang et al., 2002, 2003). As shown in
Fig. 5, SNAREs are needed for Ca2! release at a stage during
or after Ypt7p function, for example, during docking. In ad-
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Docking-dependent Ca2! release occurs through
a novel pathway
To further characterize the nature of docking-dependent
Ca2! release, we tested vacuoles derived from several mutants with defects in Ca2! transport. Deletion of the genes
encoding each of the three known Ca 2! channel subunits
in budding yeast, Cch1p, Mid1p (Fig. 9, A and B), and
Yvc1p (not depicted), did not substantially change docking-dependent Ca2! release. Pmc1p encodes a vacuolar
Ca2! ATPase that binds and is regulated by the R-SNARE
Nyv1p (Takita et al., 2001), suggesting that it might be
involved in SNARE-mediated Ca2! fluxes. Vacuoles from
pmc1% cells indeed sequestered Ca 2! slowly, resulting in a
“resting” Ca2! level elevated by !200 nM when docking
was prevented (Fig. 9 A). However, when docking was allowed to proceed a docking-dependent Ca 2! efflux of normal amplitude was observed (Fig. 9 B). These results suggest that Pmc1p is not needed for docking-dependent
Ca 2! efflux. However, it is possible that the dockingdependent flux occurs both through SNARE-mediated
inactivation of Pmc1p and opening of a channel. Therefore, we constructed double mutants containing the
pmc1% allele in combination with cch1% or mid1% alleles.
Vacuoles from each of these double mutant strains exhibited Ca2! sequestration and efflux phenotypes similar to
the pmc1% single mutant (Fig. 9, A and B). These results
suggest that docking-dependent Ca2! release from the
vacuole occurs through a novel pathway.
Discussion
Published January 20, 2004
Ca2! and SNAREs | Merz and Wickner 203
Figure 10. Current working model of SNARE and Ca2! dynamics
on the yeast vacuole. See text for discussion.
Role of Sec17/18p
Two types of staging experiments, Ypt7p block release and
Vam7p bypass of Sec17/18p function, indicate that Sec17/
18p need not function during the Ca2! release event itself.
Instead, Sec17/18p are required for sustained cycles of Ca2!
release, as shown in Fig. 5 B, Fig. 6 B, and Fig. 7 A. Sec17/
18p catalyze the disassembly of SNARE complexes, liberating unpaired SNAREs which can participate in new trans interactions. Recombinant Sec18p promotes Ca2! release with
maximal stimulation at !300 nM (Fig. 5 A), close to the in
vivo level of !200 nM (Ghaemmaghami et al., 2003). At
higher levels of Sec18p ('400 nM), the stimulation of Ca2!
release is attenuated, possibly due to the disassembly of
trans-SNARE complexes (Ungermann et al., 1998b) before
they can complete their stimulation of Ca2! efflux. When
Sec17/18p function is prevented, Ca2! release decays rapidly. These observations suggest that each trans-SNARE interaction event triggers only a short-lived transient of Ca2!
release, and that repeated cycles of trans-SNARE interactions are needed for sustained Ca2! release.
The Ca2! release pathway
Our data show that docking-dependent trans-SNARE interactions promote Ca2! release. However, we emphasize that
the functional trans-SNARE interactions defined in this paper might not be identical to SNARE core complexes or
trans-SNARE complexes isolated from vacuole detergent extracts (Ungermann et al., 1998b); additional work is needed
Implications of SNARE-dependent Ca2! release
during docking
The physical proximity of Ca2! channels to the fusion machinery is suggested by experiments in which fusion is prevented by fast, but not slow, Ca2! chelators (Sullivan et al.,
1993; Neher, 1998; Peters and Mayer, 1998; Pryor et al.,
2000), and by the brief interval ((200 $s) between channel
gating and exocytosis in neurons (Llinas et al., 1981). Furthermore, many reports document physical and regulatory
interactions between Ca2! channels and SNARE proteins in
animal cells (Bennett et al., 1992; Yoshida et al., 1992;
Sheng et al., 1994; Mochida et al., 1996; Wiser et al., 1996;
Rettig et al., 1997). Ca2! channels associate with other fusion factors, including Rab3-interactor binding proteins
(Hibino et al., 2002) and the synaptic Ca2! sensor synaptotagmin (Sudhof, 2002). SNAREs are also implicated in
store-operated Ca2! entry, which may require membrane
docking (Yao et al., 1999). Interactions between Ca2! signaling proteins and docking and fusion factors could have
two functions: to allow channels to monitor the functional
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dition, Rho GTPases and the SM-protein Vps33p act at
docking, regulate SNARE complex formation (Sato et al.,
2000; Eitzen et al., 2001; Muller et al., 2001), and are
needed for Ca2! release.
The most direct evidence that trans-SNARE interactions
trigger Ca2! release is shown in Fig. 4: in vitro complementation between populations of vacuoles lacking either
Vam3p or Nyv1p indicates that these SNAREs can act from
opposite membranes to trigger docking-dependent Ca2! release. Moreover, rVam7p stimulation of Ca2! release requires both Vam3p and Nyv1p, even when these SNAREs
are located in trans to one another (Fig. 7 B).
to understand SNARE association cycles and dynamics.
Docking-dependent Ca2! release may be triggered directly
by SNARE interactions with a Ca2!-releasing channel, or release may result from sequential interactions of SNAREs on
apposed membranes with unidentified intermediary molecules.
It is unlikely that the observed Ca2! release occurs as an
indirect consequence of fusion events. Mayer’s group recently reported that vacuoles isolated from vph1% cells dock
and release Ca2! in a Ypt7p-dependent manner, but do not
go on to fuse (Bayer et al., 2003). In addition, manipulation
of Sec17/18p function after priming can dramatically raise
or lower the amplitude of Ca2! release with only modest effects on fusion. These observations, together with the Ca2!dependence of fusion itself (Peters and Mayer, 1998),
strongly suggest that the Ca2! release events studied here occur during docking, not after fusion.
A channel or transporter required for docking-dependent
Ca2! release from the vacuole has not yet been identified,
despite our efforts (Fig. 8) and studies of vacuolar Ca2! homeostasis in other laboratories (Takita et al., 2001; Bayer et
al., 2003; Zhou et al., 2003). A number of uncharacterized
ORFs in S. cerevisiae appear to encode ion transporters, and
one of these could be responsible for docking-dependent
Ca2! release. However, redundancy among more than one
transporter might frustrate efforts to identify the relevant
proteins through analyses of single knockouts.
Docking-dependent Ca2! release might occur through less
conventional mechanisms. In most models of fusion, lipids
at the fusion site transiently assume nonbilayer morphologies. Simulations (Muller et al., 2003) indicate that these rearrangements may form transient pores between cytoplasmic
and noncytoplasmic compartments, and careful measurements of fusion events mediated by the influenza hemagglutinin protein confirm that transient leakage currents can
accompany fusion (Frolov et al., 2003). Trans-SNARE
complex formation might directly promote ion flux by perturbing bilayer structure.
Published January 20, 2004
204 The Journal of Cell Biology | Volume 164, Number 2, 2004
status of docking over time, and to ensure that the fusion
machinery and regions of peak Ca2! flux coincide in space
(Neher, 1998). For intracellular fusion events, these interactions may trigger Ca2! flux in response to successful docking. In synapses, where voltage-gated Ca2! channels respond
to membrane depolarization, similar mechanisms might bias
Ca2! flux toward channels associated with primed and
docked vesicles. Our experiments with vacuoles suggest that
trans-SNARE complex formation is a checkpoint that controls progression to fusion. In this view, trans-SNARE interactions signify that docked membranes reside within a certain minimum distance and verify that specific biochemical
events have transpired (e.g., priming and vertex subdomain
assembly), triggering Ca2! release and downstream events
leading to fusion.
Materials and methods
Yeast strains
Reagents
rVam7p (residues 2–316) and Vam7p PX domain (residues 2–123) were
expressed as GST fusions from the pGEX-KT vector (Hakes and Dixon,
1992) in BL21-pRP cells (Stratagene). VAM7 sequences were amplified
from BJ3505 DNA using a forward primer with an engineered BamH1 site
(5*-cgcGGATCCGCAgctaattctgtaggg-3*) and reverse primers with engineered EcoR1 sites (5*-cgGAATTCTCAagcactgttgttaaaatgtctagc-3* for
rVam7p, and 5*-cgGAATTCACTTtgacaactgcaggaagac-3* for PX). Cells
were grown in TB medium (Maniatis et al., 2001), 200 mg/l ampicillin, and
34 mg/l chloramphenicol to OD600 ) 2.3, and expression was induced
with 0.5 mM IPTG for 4 h at 26#C. Cell pellets (10,000 g, 20#C, 5 min)
were resuspended in two pellet volumes of PBS with 2 mM EGTA, 1 mM
EDTA, 1+ protease inhibitor cocktail (Haas, 1995), 1 mM PMSF, and
0.01% 2-mercaptoethanol. The cell suspension was frozen dropwise in
liquid N2 and stored at –80#C. Cells were thawed, lysed in a French press,
mixed with Triton X-100 (0.5% final), and rocked for 20 min at 4#C before
clearing (20,000 g, 4#C, 30 min). Glutathione-agarose (1 ml of slurry per
10 ml of lysate; Amersham Biosciences) was added to cleared lysate and
rocked overnight at 4#C. The beads were washed with PBS supplemented
as above (20 bed volumes), then with PBS alone (20 bed volumes), and finally with cleavage buffer (50 mM TrisCl, pH 8.0, 100 mM NaCl, 2 mM
CaCl2, 0.1% 2-mercaptoethanol; 10 bed volumes). Proteins were cleaved
from the support with thrombin (!10 U/mg fusion protein, in two to three
bed volumes of cleavage buffer; Sigma-Aldrich) for 3–4 h at 23#C. Thrombin was removed by flowing the eluted cleavage products directly over a
second column containing 1 ml p-aminobenzamidine–agarose (SigmaAldrich). Proteins were exchanged into reaction buffer (20 mM PIPES/
KOH, pH 6.8, 125 mM KCl, 5 mM MgCl2, 200 mM sorbitol) on G-25 resin
(Amersham Biosciences). Aliquots were frozen in liquid N2, stored at
–80#C, and thawed just before use.
Antibodies were raised against peptides from Ypt7p (Eitzen et al., 2001),
Vps33p (for Vps33p, NH2-CLEDTEQWQKDGFDLNSKKT, and NH2-CIEDEHAADKITNENDDFSEA); against rVam7p and Sec17p (Haas and Wickner, 1996); and against recombinant cytoplasmic domains of Vam3p
(Nichols et al., 1997), Nyv1p (Ungermann et al., 1998a), and Vti1p (Ungermann et al., 1999). IgG or affinity-purified antibodies were prepared as
Fusion
30 $l of standard fusion reactions contained a 1:1 mixture of vacuoles
(Haas, 1995) from strains BJ3505 and DKY6281 (6 $g total protein by
Bradford assay; premixed in PS buffer) added to a mixture containing: 20
mM Pipes/KOH, pH 6.8, 200 mM sorbitol, 125 mM KCl, 5 mM MgCl2, 10
$M coenzyme A, 4.5 $M recombinant Pbi2p (IB2), 10 nM recombinant
His6-Sec18p (except as noted), 1% (wt/vol) defatted BSA (Sigma-Aldrich),
and 0.03+ protease inhibitor cocktail (Haas, 1995). ATP-regenerating system (Haas, 1995) was added from a 10+ stock to 1+ final (0.67+ for Ca2!
efflux assays). Fusion assays were incubated for 80 min at 27#C or as noted
in figure legends, and fusion was measured by determining the amount of
active alkaline phosphatase as described previously (Haas, 1995) except
that phosphatase assay buffer was supplemented with 10 mM CaCl2. Ca2!
efflux assays were performed with 10 $g rather than 6 $g of vacuoles per
30 $l vol. For experiments using vacuoles from mutant cells, highly pure
oxalyticase (Enzogenetics) was used to prepare spheroplasts for vacuole
isolation.
Ca2! assay
Aequorin luminescence assays were performed as described previously
(Peters and Mayer, 1998; Muller et al., 2002) with modifications. Samples
were analyzed in 96-well, low protein binding, conical bottom plates
(Nunc) in a luminometer (Molecular Devices). IGOR Pro 4 (WaveMetrics)
and JMP 5 (SAS Institute) were used for data analysis. Calibration was done
using buffered Ca2! EGTA standard solutions containing reaction salts including the ATP regenerating system, prepared as described previously
(Tsien and Pozzan, 1989), and checked with MAX Chelator (Bers et al.,
1994) version WEBMAXC 10.14.2002 (http://www.stanford.edu/~cpatton).
Commercial Ca2! standards gave similar results. A curve was fit to the luminance for each Ca2! standard divided by peak luminance at saturating
Ca2! (L/Lmax), using a two-state model with three Ca2!-binding sites (Allen
et al., 1977): L/Lmax ) [(1 ! KR [Ca2!])/(1 ! KTR ! KR [Ca2!])]3.
We obtained KR ) 3.88 , 0.88 + 106 M-1 and KTR ) 1.45 , 0.29 +
102 (mean , SD) similar to values reported by Allen et al. (1977). KR and
KTR were used to calculate [Ca2!] in experimental samples. In addition to
Ca2!-dependent luminance, aequorin undergoes irreversible, Ca2!-dependent inactivation. At constant-free [Ca2!] (i.e., in buffered standard solutions), the apparent [Ca2!] gradually decays exponentially over time. At
640 nM of free [Ca2!], the decay had a time constant . ) 95 , 4 min
(mean r2 ' 0.97). The traces shown are not adjusted for aequorin inactivation.
Microscopy
Images were obtained on a microscope (model BX51/61; Olympus) with a
Hg arc lamp, a 60+ 1.4 NA Plan Apochromat objective, and a camera
(model Sensicam QE; Cooke). The camera and microscope are controlled
by IP Lab (Scanalytics). Vacuole sizes were measured by photographing
wet mounts of reactions containing 2 $M MDY-64, a fluorescent lipid
probe (Molecular Probes). Vacuoles were measured using Image/J (http://
rsb.info.nih.gov/ij/) by manually circumscribing vacuoles with best-fit ellipses. The mean of the major and minor diameters of each ellipse was
used to estimate vacuole surface area, by approximating vacuoles as
spheres of radius diam/2. Surface areas were distributed approximately log
normally, so these data were log transformed before statistical analysis (in
JMP 5; SAS Institute) and geometric means were reported.
Online supplemental material
Video 1. Video microscopy of fusion occurring after rVam7p rescue of an
anti-Sec17p block. Video microscopy images were acquired with a 60+
1.4 NA Plan Apochromat objective and a camera (model Sensicam QE;
Cooke) mounted on a hybrid BX51/61 platform (Olympus). The camera
and microscope were controlled by IP Lab (Scanalytics) running under
Mac OS X. Illumination was provided by an Hg arc lamp (Olympus).
Time-lapse observations were made under illumination attenuated by
'98% using neutral density filters (Olympus) and an infrared-blocking filter (Schott), and data was acquired with 2 + 2 pixel binning. Images were
acquired at ambient temperature, measured at 23–25#C with a thermocouple on the microscope stage. For time-lapse sequences a sharpening filter
Downloaded from jcb.rupress.org on April 4, 2011
The standard strains used in our assays are BJ3505 (MAT" pep4::HIS3
prb1-%1.6R his3–200 lys2–801 trp1-%101 [gal3] ura3–52 gal2 can1;
Jones, 2002) and DKY6281 (MAT" leu2–3 leu2–112 ura3–52 his3-%200
trp1-%901 lys2–801; Haas et al., 1994). vam3% and nyv1% derivatives of
BJ3505 and DKY 6281 were prepared as described previously (Nichols et
al., 1997). BY4742 (MAT" his3%1 leu2%0 lys2%0 ura3%0; Brachmann et
al., 1998), BY4742 yvc1%::neo, BY4742 cch1%::neo, and BY4742 mid1%::
neo were obtained from Research Genetics. BY4742 and its derivatives
were used to generate AMY10 () BY4742 pmc1%::LEU2), AMY11 ()
BY4742 cch1%::neo pmc1%::LEU2), and AMY12 () BY4742 mid1%::neo
pmc1%::LEU2) by transformation with HindIII-cut pKC52 (Cunningham
and Fink, 1994) and selection on CSM lacking Trp. The double mutant
strains AMY11 and AMY12 grew well on YPD medium (Difco) and had
similar growth defects as the pmc1% single mutant AMY10 on YPD medium with 0.2 M Ca2!.
described previously (Harlow and Lane, 1999). For anti-Ypt7p reversal
(Eitzen et al., 2001) peptide was used at 20 $g/ml final. Antibodies were
stored in PS buffer (20 mM Pipes/KOH, 200 mM sorbitol, pH 6.8) at 4#C or
–80#C after buffer exchange on Sepharose G-25 resin (Amersham Biosciences) or dialysis. Some antibody preparations were concentrated in
Microcon-30 ultrafiltration devices (Millipore).
Published January 20, 2004
Ca2! and SNAREs | Merz and Wickner 205
(5 + 5 hat) was applied to each frame. Online supplemental material is
available at htttp://www.jcb.org/cgi/content/full/jcb.200310105/DC1.
This work was funded by the National Institute of General Medical Sciences. A.J. Merz gratefully acknowledges support from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR07576-09) and
the Damon Runyon Cancer Research Foundation (DRG-1598).
Submitted: 22 October 2003
Accepted: 4 December 2003
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